Analysis and Therapy of Age-Dependent Proteostasis Failure in Neurodegeneration

神经变性中年龄依赖性蛋白质稳态失败的分析和治疗

• 批准号: 8666527
• 负责人: Robert Joseph Shmookler Reis
• 金额: --
• 依托单位: CENTRAL ARKANSAS VETERANS HLTHCARE SYS
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8666527
• 关键词: Acetylation; Acylation; Adherence; Affect; Age; Alzheimer' s Disease; Amyloid; Amyotrophic Lateral Sclerosis; Animal Model; Antibodies; Antioxidants; Appearance; Asses; Ataxia; Autophagocytosis; Biological Models; Brain; Caenorhabditis elegans; Cell Respiration; Cells; Central Nervous System Part; Chemical Agents; Chemicals; Cleaved cell; Clinical; Codon Nucleotides; Complex; Craniocerebral Trauma; Defect; Dementia; Detergents; Diagnostic; Disease; Drug Metabolic Detoxication; Elderly; Equilibrium; Exposure to; Failure; Familial disease; Generations; Genes; Genetic; Health; Human; Huntington Disease; Individual; Ions; Label; Lead; Learning; Length; Lesion; Link; MAPK8 gene; Maintenance; Mitochondria; Modeling; Modification; Molecular; Molecular Chaperones; Mus; Mutation; Nematoda; Nerve Degeneration; Neurodegenerative Disorders; Neurofibrillary Tangles; Neurons; Neuropathy; Neurotoxins; Parkinson Disease; Parkinson' s Dementia; Pathway interactions; Patients; Peptide Fragments; Peptides; Pharmaceutical Preparations; Phosphorylation; Play; Post-Translational Protein Processing; Procedures; Process; Property; Proteins; Psyche structure; RNA Interference; Recycling; Relative (related person); Reporting; Research Proposals; Risk; Role; Sampling; Scanning; Severities; Signal Pathway; Site; Specificity; Stable Isotope Labeling; Structural Protein; Structure; Superoxide Dismutase; System; Temperature; Testing; Therapeutic; Tissue Sample; Tissues; Toxic Environmental Substances; Toxic effect; Toxicant exposure; Transgenes; Transgenic Organisms; Trinucleotide Repeats; Triplet Multiple Birth; Trypsin; Ubiquitin; Validation; Veterans; Viral; age related; base; brain cell; catalase; clinical Diagnosis; clinically relevant; comparative; crosslink; design; gel electrophoresis; glycation; human FRAP1 protein; improved; in vivo; mind control; multicatalytic endopeptidase complex; neuropathology; neurotoxic; neurotoxicity; oxidation; oxidative damage; polyglutamine; prevent; protein TDP-43; protein aggregate; protein aggregation; protein complex; protein misfolding; repaired; repository; synuclein; tau Proteins; ubiquilin

DESCRIPTION (provided by applicant): Most major neurodegenerative diseases are characterized by the formation of toxic protein aggregates which compromise a specific part of the central nervous system. Clinical diagnosis has been guided by the regions affected, but it is becoming increasingly evident that the basic processes underlying Alzheimer dementia are remarkably similar to those leading to Parkinson's and Huntington's diseases, and Amyotrophic Lateral Sclerosis. Although all cells are obliged to maintain proteostatic equilibrium, neurons are at particular risk due to high rates of oxidative metabolism. Specific genetic lesions (or possibly chemical or viral exposure, in sporadic cases) may determine the site of neuropathy as the "weakest link" based on the balance of pro- aggregation and countervailing factors. Factors favoring aggregation include high local abundance of vulnerable proteins and structural instability (determined by the native folded structure, exposed reactive groups, and triplet-array length, if present), oxidative damage, glycation, and protein-reactive metabolites or environmental toxicants that produce cross-linking or adduction. Protective factors include antioxidant defenses (SODs, GSTs, catalases, etc.), detoxification/clearance systems, levels of HSP/chaperones to refold transiently misfolded proteins (preventing their adherence to aggregates via exposed hydrophobic residues), protein repair of covalent molecular damage that would favor misfolding, and proteasomal degradation of ubiquitinylated proteins. Autophagy may be able to clear the large insoluble aggregates diagnostic of these neuropathologies, but it remains controversial whether those are indeed the most neurotoxic complexes, or are instead less-toxic, sequestered repositories formed from smaller, soluble aggregates which are more potent neurotoxins. This issue could be resolved through a careful, comparative analysis of soluble vs. insoluble aggregates, "seeded" by several of the diverse initiator proteins observed in neuropathies, defining proteins and post-translational modifications in C. elegans and human neuropathology samples. The current proposal has three intertwined and complementary aims: (1.) To define proteins that aggregate in C. elegans models of Alzheimer's, initiated or seeded by neuron-specific tau and Ass transgenes. Protein complexes from nematodes with age-dependent or induced aggregates will be pulled down with antibody to tau or Ass, and then separated into detergent-insoluble and soluble fractions. Proteins from each fraction will be resolved by gel electrophoresis, and peptides will be cleaved by trypsin and analyzed by LC-MS2 to define and quantify the proteins and their post-synthetic modifications. The most sensitive and reliable quantitation uses product-ion scans to seek diagnostic fragments from predicted peptides; in parallel, we will also conduct more general but less sensitive "unbiased scans" to identify and quantify the more abundant proteins, even if unforeseen. Results will be cross-checked and extended by unrestrictive searches to quantify postsynthetic modifications including phosphorylation, acetylation, oxidation, acylation, glycation, etc., and thus implicate mechanisms involved in their age-dependent occurrence. Dual-label procedures will allow robust relative quantitation, and the validation of proteins that aggregated in vivo rather than during handling. (2.) To seek similar aggregates in human samples. De-identified samples from affected and control brain sites from AD subjects will be studied along with comparable-site samples from normal controls, and their aggregates immunoprecipitated with antibodies to Ass and tau. Complexes will be fractionated and separated as for Aim 1, and aggregates subjected to LC-MS2 analyses as above, seeking protein markers or modifications including any were implicated in the C. elegans models. (3.) To assess which implicated proteins and modifications play functional roles in neurotoxicity, it will be made worse by RNA interference targeting protective genes, or relieved by therapeutic drugs or RNAi against pro-aggregative genes. Proteins or modifications in either soluble or insoluble aggregates, which vary concordantly with neurotoxicity, become functional candidates

描述（由申请人提供）： 大多数主要的神经退行性疾病的特征在于形成毒性蛋白质聚集体，其损害中枢神经系统的特定部分。临床诊断一直以受影响的区域为指导，但越来越明显的是，阿尔茨海默氏痴呆症的基本过程与导致帕金森病和亨廷顿病以及肌萎缩性侧索硬化症的基本过程非常相似。尽管所有细胞都必须维持蛋白质稳定平衡，但由于氧化代谢的高速率，神经元处于特别危险的状态。特定的遗传性病变（或可能的化学或病毒暴露，在散发的情况下）可能会决定神经病变的网站作为“最薄弱的环节”的基础上的平衡，促聚集和抵消因素。有利于聚集的因素包括脆弱蛋白质的高局部丰度和结构不稳定性（由天然折叠结构、暴露的反应基团和三联体阵列长度（如果存在）确定）、氧化损伤、糖化和产生交联或内收的蛋白质反应性代谢物或环境毒物。保护因素包括抗氧化防御（SOD、GST、过氧化氢酶等），解毒/清除系统，HSP/伴侣蛋白的水平以再折叠瞬时错误折叠的蛋白（防止它们通过暴露的疏水残基粘附到聚集体），有利于错误折叠的共价分子损伤的蛋白修复，以及泛素化蛋白的蛋白酶体降解。自噬可能能够清除诊断这些神经病理学的大的不溶性聚集体，但仍然存在争议，这些聚集体是否确实是最具神经毒性的复合物，或者是毒性较小的隔离储存库，由较小的可溶性聚集体形成，这些聚集体是更有效的神经毒素。这个问题可以通过对可溶性与不溶性聚集体进行仔细的比较分析来解决，这些聚集体被神经病中观察到的几种不同的起始蛋白“接种”，定义了C中的蛋白和翻译后修饰。线虫和人类神经病理学样本。目前的建议有三个相互交织和相辅相成的目标：（1）。为了确定聚集在C. elegans阿尔茨海默氏症模型，由神经元特异性tau和Ass转基因启动或播种。来自线虫的具有年龄依赖性或诱导的聚集体的蛋白质复合物将用tau或Ass的抗体拉下，然后分离成洗涤剂不溶性和可溶性级分。将通过凝胶电泳分离各组分中的蛋白质，并通过胰蛋白酶切割肽，并通过LC-MS 2进行分析，以确定和定量蛋白质及其合成后修饰。最灵敏和可靠的定量使用产物离子扫描来从预测的肽中寻找诊断片段;同时，我们还将进行更一般但不太灵敏的“无偏扫描”来识别和定量更丰富的蛋白质，即使是不可预见的。将通过非限制性检索对结果进行交叉检查和扩展，以定量合成后修饰，包括磷酸化、乙酰化、氧化、酰化、糖化等，从而揭示了 年龄依赖的发生。双标记程序将允许稳健的相对定量，并验证在体内而不是在处理过程中聚集的蛋白质。（2.）在人体样本中寻找相似的聚集体。来自AD受试者的受影响和对照脑部位的去识别样本将与来自正常对照的可比部位样本一起进行沿着研究，并且其聚集体用针对Ass和tau的抗体进行免疫沉淀。如目的1所述，将复合物分级并分离，并对聚集体进行如上所述的LC-MS 2分析，寻找蛋白质标记物或修饰，包括任何与C. elegans模型（3.）为了评估哪些相关蛋白质和修饰在神经毒性中发挥功能作用，靶向保护性基因的RNA干扰将使其恶化，或通过治疗药物或针对促聚集基因的RNAi缓解。可溶性或不溶性聚集体中的蛋白质或修饰，其变化与神经毒性一致，成为功能候选物